Microbial pathogens that inhabit our environment must undergo a radical change to survive inside a mammalian host. Among the more than 100,000 different species of environmental fungi are six phylogenetically related ascomycetes called the dimorphic fungi: Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenkii, and Penicillium marneffei. These fungi change morphology once spores are inhaled into the lungs of a mammalian host from hyphal molds in the environment to pathogenic yeast forms. Dimorphic fungi inhabit the soil worldwide and collectively cause over a million new infections a year in the United States alone. They tend to remain latent after infection and may reactivate if the subject becomes immune-deficient (J. N. Galgiani, Ann Intern Med 130, 293 (1999); L. Ajello, Distribution of Histoplasma capsulatum in the United States. C. W. Ajello L, Furcolow M F, Ed., Histoplasmosis (Charles C. Thomas Publishers, Springfield Ill., 1971), pp. 103-22; L. J. Wheat et al., Medicine (Baltimore) 69, 361 (1990); T. M. Chiller, J. N. Galgiani, D. A. Stevens, Infect Dis Clin North Am 17, 41 (Mar, 2003); B. S. Klein, J. M. Vergeront, J. P. Davis, Semin Respir Infect 1, 29 (1986).). It has long been believed that phase transition from mold to yeast is obligatory for pathogenicity, but the mechanism that regulates this switch has remained a mystery. In this report, we provide firm genetic evidence that establishes the central role of dimorphism in pathogenicity, and describe a regulator of this morphologic transition.
It is temperature that induces dimorphic fungi to change phases (B. Maresca, G. S. Kobayashi, Microbiol Rev 53, 186 (1989).). At 25° C., they grow as mold. At 37° C., the core temperature of humans, they switch into the pathogenic yeast form (G. Medoff et al., Science 231, 476 (1986).), during which yeast-phase specific virulence genes are induced. Few of these genes have been identified; among the best studied are BAD1 of B. dermatitidis, CBP1 of H. capsulatum and the α-(1,3)-glucan synthase (AGS1) of these fungi and P. brasiliensis (B. S. Klein, J. M. Jones, J Clin Invest 85, 152 (1990); T. S. Sebghati, J. T. Engle, W. E. Goldman, Science 290, 1368 (2000); L. H. Hogan, B. S. Klein, Infect Immun 62, 3543 (1994).). We postulated that deciphering the regulation of phase-specific genes would elucidate the control of morphogenesis.
Forward genetics, a process of inducing mutations randomly in a genome to detect phenotypes and linked genes, has advanced our understanding of microbial pathogenesis. Dimorphic fungi have not yet been manipulated in this way because the classical genetic approaches have proved too cumbersome and the molecular tools have been unavailable. We previously showed that Agrobacterium tumefaciens transfers DNA randomly into the genomes of B. dermatitidis and H. capsulatum, primarily into single sites and without recombination, in theory, allowing the identification of recessive mutations (T. D. Sullivan, P. J. Rooney, B. S. Klein, Eukaryot Cell 1, 895 (2002).). In the present invention, we disclose the use of A. tumefaciens for insertional mutagenesis in a dimorphic fungus to attempt to uncover regulators of yeast-phase specific genes and phase transition from mold to yeast.